Adenoid Cystic Carcinomas (ACCs) represent one of the most common and biologically aggressive forms of epithelial malignancy of the salivary glands, for which there are no clinically approved treatments. ACCs are characterized by a high tendency towards peri-neural infiltration and distant-site metastasis, and are largely refractory to conventional chemo-radiotherapy approaches. ACCs are also defined by a distinctive biological feature: the co-existence within malignant tissues of two phenotypically distinct sub-populations of cancer cells (ductal-like vs. myoepithelial-like) characterized by profoundly distinct gene-expression profiles and functional properties. This study will leverage a portfolio of surface markers recently identified by our laboratory that allow, for the first time, the differential purification, molecular study and functional analysis of these two cell types. The hypotheses formally tested by this study are: a) that the two cell types co-exist in a dynamic and hierarchical equilibrium, whereby one cell type functions as a progenitor for the other; and b) that biochemical pathways regulating the conversion of one cell type into the other can be leveraged to develop new therapeutic approaches against ACCs. The project envisions two specific aims: 1) to elucidate the developmental relationship between the two cellular components of ACCs: we will use fluorescence activated cell sorting (FACS) to purify the two sub-populations of cancer cells from human ACCs, and then employ prospective in vivo xenotransplantation experiments and single-cell RNA-seq technologies to test whether the two cell types: a) display robust and systematic differences in tumor-initiating capacity across patient-derived xenograft (PDX) lines from independent patients; b) are hierarchically related (i.e. whether both can plastically inter-covert into each other or whether one serves as the progenitor of the other, in a mono-directional fashion); c) contain additional, previously unrecognized cellular sub-populations, themselves characterized by distinct molecular and functional properties; 2) to test the in vitro and in vivo therapeutic efficacy of novel drug combinations against ACCs, designed to target biochemical pathways shown to affect the differential representation and preferential survival of the two cellular sub-types: we will treat 3D organoids and PDX lines shown to recapitulate the dual cell composition of primary ACCs with sequential drug regimens, designed to, in a first step, induce the differentiation of myoepithelial-like cells into ductal-like cells and, in a second step, to selectively eliminate ductal-like cells; such novel drug regimens will be tested for their capacity to: a) cause changes in the cell composition of malignant tissues (ductal/myoepithelial cell ratio); b) reduce tumor size and/or growth kinetics; c) prevent in vivo metastatic dissemination (using non-invasive bio-luminescent reporters). Significance. This study will elucidate the molecular and functional properties of the two cell types found in human ACCs, filling a major scientific gap in salivary tumor biology, and addressing an unmet clinical need in salivary gland oncology: the identification of novel drug targets for the pharmacological treatment of ACCs.